Cell differentiation protocols to enhance the expression of drug metabolizing enzymes in BFH12 cells: are they effective?

In vitro culture of primary hepatocytes represents the preferred platform for drug development, toxicology and drug-drug interaction studies. Cattle hepatocytes have already been successfully used to investigate xenobiotic metabolism, but they suffer from the same limitations as its human counterpart, specifically a time-dependent reduction of expression and biological activity of drug metabolizing enzymes (DMEs). Additionally, cattle hepatocytes rapidly encounter apoptotic phenomena if the liver is sampled 30 minutes after the sacrifice and the bleeding step of the animal. Since there are no adult bovine hepatoma cell lines, and with the aim of overcoming the disadvantages of bovine primary hepatocytes, a recent immortalized foetal hepatocyte cell line (BFH12) has been established1. We recently characterized this cell model; we showed a good responsiveness of BFH12 cells towards an aryl hydrocarbon receptor (AHR) agonist, PCB1262, and a slight expression of pregnane X receptor (PXR), CYP3A38 and CYP3A48, associated to a low responsiveness to prototypical CYP3A inducers3. Therefore, the present study aimed to test different human hepatic differentiation protocols by adding known differentiation inducers to the culture medium, and/or extending the maintenance time, to enhance the mRNA expression of CYP1As and CYP3As and the main nuclear receptors (NRs) involved in their regulation. Specifically, we focused on the mRNA expression of CYP1A1, CYP1A2, CYP3A28, CYP3A38 and CY3A48, as well as their regulators, AHR, PXR, constitutive androstane receptor (CAR), and retinoid X receptor (RXR). BFH12 cells were maintained under 4 different culture conditions: standard medium4with 1% glutamine for 4 weeks (A); standard medium (i.e., condition A) plus 1% nonessential amino acids, and 10 µM HEPES for 1 week (B), then supplemented with 1% DMSO, 1µM DEX, 10µM hydrocortisone and insulin for an additional week (C) or 1% DMSO only for further 2 weeks5 (D). Then, mRNA expression of genes of interest was measured by means of Real-time PCR. Culture condition A enhanced the expression of all four target NRs, even if the statistical significance (P< .01) both CYP1A1 and CYP3A28 mRNAs. Overall, our findings showed that the most effective differentiation protocols in BFH12 cells were the maintenance of standard culture conditions for one month (i.e., condition A) and the addition of 1% non-essential amino acids and 10 µM HEPES for 1 week (i.e., condition B). Nevertheless, the protocols here used and adapted from human hepatoma cell lines were only partially effective on BFH12 cells, thus supporting the previously observed species-differences between human and cattle in hepatic DMEs regulation, as well as the potential ontogenetic effects (foetus versus adult).